Diagnostic assays have become an indispensable means for detecting analytes in test samples by using the mutual reaction between the analyte and a specific binding member for the analyte, such as the immunoreaction between an antigen and the antibody to that antigen. Typically, detectable tags or labels attached to antibodies, which in turn bind to the analyte of interest, are employed in such diagnostic assays, wherein the detection of the resultant labeled antibody/analyte complex, or of the labeled antibody which remains unbound, is used to indicate the presence or amount of analyte in the test sample.
Two commonly used binding assay techniques are the radioimmunoassay (RIA) and the enzyme immunoassay (EIA), both of which employ a labeled binding member. The RIA uses a radioactive isotope as the detectable tag or label attached to a binding member. Because the radioactive isotope can be detected in very small amounts, it can be used to detect or quantitate small amounts of analyte. However, substantial disadvantages associated with the RIA include the special facilities and extreme caution that are required in handling radioactive materials, the high costs of such reagents, and their unique disposal requirements.
The EIA uses an enzyme as the detectable tag or label attached to a binding member, wherein the enzymatic activity of the enzyme is used to detect the immunoreaction. While the EIA does not have the same disadvantages as the RIA, EIA techniques typically require the addition of substrate materials to elicit the detectable enzyme reaction. Enzyme substrates are also often unstable and have to be prepared just prior to use or be stored under refrigeration. In addition, enzyme labels may be difficult to purify and conjugate to binding members, and may be unstable during storage at room temperature. Enzyme immunoassays are also unsatisfactory in that the methods typically require complex incubations, multiple liquid additions and multiple wash steps. Moreover, even under refrigerated conditions, enzymes are unstable.
More recently, assay techniques using metallic sol particles as visual labels have been developed. In these techniques, a metal (e.g., gold, silver, platinum), a metal compound, or a nonmetallic substance coated with a metal or a metal compound, is used to form an aqueous dispersion of particles. Generally, the binding member to be labeled is coated onto the metal sol particles by adsorption, and the particles are captured or aggregated in the presence of analyte. Although the metal sol particles have the advantage of producing a signal that is visually detectable as well as measurable by an instrument, they, nevertheless, are difficult to quantitatively measure. The metallic particles also have a limited color intensity, and therefore limited sensitivity in some assays. In addition, the surfaces of inorganic metallic colloid particles, such as gold, do not readily accept the covalent attachment of binding members. Thus, during use in a binding assay, care must be taken so that the adsorbed binding members are not removed from the inorganic particles through the combination of displacement by other proteins or surface active agents and the shear forces which accompany washing steps used to remove non-specifically bound material. Sol particles can be difficult to coat without inducing aggregation, may aggregate upon storage and may aggregate upon the addition of buffers or salts. Furthermore, such particulate labels are difficult to concentrate, may aggregate during use and are difficult to disperse.
Other label materials include chemiluminescent and fluorescent substances. Non-metallic particles, such as dyed or colored latex and selenium particles have also been used as visual labels.
Prior to the present invention, magnetic particles and magnetic fields have generally been used as means to remove or position an analyte component of a test sample. For example, U.S. Pat. Nos. 4,070,246 and 3,985,649 describe the use of binding members attached to ferromagnetic particles, whereby the binding member forms a complex with the analyte of interest, and the resulting complex is removed from the reaction mixture by means of a magnetic field. Alternatively, U.S. Pat. No. 3,933,997 describes the use of magnetic particles and a magnetic field as a means of concentrating a radioactive material on a test substance. U.S. Pat. No. 4,219,335 describes the use of magnetic particles which have characteristics capable of affecting electrical resistance, wherein a capacitance measurement will reveal whether the particles are present on a surface. However, the effect of the magnetic field on the magnetic particles has no relation to the presence or amount of analyte in the test sample.